The biodistribution characteristics of a humanized anti-Lewis(y) antibody (hu3S193) radiolabeled to three radioisotopes, 125I, 111In, and 90Y, were examined in a BALB/c nude mouse xenograft model of breast cancer. The immunoreactivity of both 125I- and 111In-bound hu3S193 exceeded 50% and was 20% for 90Y. In vivo, labeled antibody was shown by gamma camera imaging and immunohistochemical and autoradiographic techniques to localize to Lewis(y)-expressing breast xenografts with minimal normal tissue uptake. Maximal radioisotope uptake peaked at 48 h for all three isotopes; however, the percentage of injected dose/gram and tumor retention were greater for 111In- and 90Y-bound antibody than for 125I-bound antibody. Although immunoreactivity of 111In- and 125I-labeled hu3S193 in serum was stable over a 5-day period, the amount of unlabeled 111In in serum was lower than 125I, which together with higher tumor uptake indicates better retention of 111In-labeled hu3S193 and catabolites within the tumor cells. Superior tumor uptake and retention of 111In-labeled hu3S193 and similar blood clearance compared with 125I-labeled hu3S193, suggest that radiometals are the preferred radioisotope for this antibody-antigen system. Humanized 3S193 is a promising new construct for the targeting and potential therapy of Lewis(y)-expressing tumors.